The delivery of nucleic acids into cells is indispensable for basic research in molecular and cell biology as well as medical applications such as gene therapy. For example, a transfection method utilizing viral vectors and a non-viral transfection method are known as a method for introducing genes into cells.
A transfection method utilizing viral vectors gives high level of gene delivery. Viral vectors are efficient carriers for gene delivery, but their use is limited by the induction of immune responses and virus-associated pathogenicity.
A non-viral transfection method does not have such problems that are found in a transfection method utilizing viral vectors and thus has the potential to produce pharmaceuticals from nucleic acids (Non-Patent Document 1). According to the non-viral transfection method, the products should be capable of being produced in large quantities with high reproducibility and acceptable cost, and stable to storage.
There are many known non-viral transfection methods including the major methods that use cationic lipids and/or cationic polymers. A series of non-viral transfection reagents containing cationic lipids or cationic polymers as main ingredients are now commercially available and provided for use. However, the transfection methods using such reagents have a problem in that the nucleic acids incorporated into cells through endocytosis are largely degraded in lysosomes resulting in low transfection efficiency. In addition, many of the commercially available transfection reagents are quite expensive at the present time, and their high costs are obstacles to experimentation with a large number and/or large scales of transfection.
The cationic polymer polyethylenimine (hereinafter, abbreviated as PEI) is an inexpensive, non-viral and non-liposomal reagent for transfection and is known to be one of the most cost-effective vehicles. PEI has a high cationic charge density potential and efficiently condenses DNA (compaction) to form stable complexes termed polyplexes that are incorporated into cells through endocytosis. Thus, an efficient transfection is achieves by using PEI (Non-Patent Document 2). In addition, PEI has a high pH-buffering capacity, leading to protection of incorporated DNA from lysosomal degradation and its efficient release from lysosomes into the cytoplasm. PEI is therefore useful for a transfection reagent, and is used to transfect mammalian cells with DNA in vitro and in vivo (Non-Patent Documents 2-4).
However, a transfection method using PEI has some problems such as unignorable degrees of toxicity and considerable deterioration in transfection efficiency due to low stability of PEI.
In order to improve transfection efficiency, extensive studies have been performed on the size, structure and chemical modification of PEI (Non-Patent Documents 4-11), and addition of supplements to a PEI/DNA polyplex or compacted DNA was also examined (Non-Patent Documents 12-13).
[Prior Arts]
[Patent Documents]
Patent Document 1: Tokuhyo 2008-509076.
Patent Document 2: Tokuhyo 2006-516259.
[Non-Patent Documents]
Non-Patent Document 1: Davis, M. E. (2002) Non-viral gene delivery systems. Curr. Opin. Biotechnol. 13, 128-131.
Non-Patent Document2: Boussif, O. et al., (1995) A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: Polyethylenimine. Proc. Natl. Acad. Sci. USA 92, 7297-7301.
Non-Patent Document 3: Abdallah, B. et al., (1996) A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: Polyethylenimine. Hum. Gene Ther. 7, 1947-1954.
Non-Patent Document 4: Goula, D. et al., (1998) Size, diffusibility and transfection performance of linear PEI/DNA complexes in the mouse central nervous system. Gene Ther. 5, 712-717.
Non-Patent Document 5: Zanta, M. A. et al., (1997) In vitro gene delivery to hepatocytes with galactosylated polyethylenimine. Bioconjug. Chem. 8, 839-844.
Non-Patent Document 6: Fischer, D. et al., (1999) A novel non-viral vector for DNA delivery based on low molecular weight, branched polyethylenimine: Effect of molecular weight on transfection efficiency and cytotoxicity. Pharm. Res. 16, 1273-1279.
Non-Patent Document 7: Ogris, M. et al., (1999) PEGylated DNA/transferrin-PEI complexes: Reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther. 6, 595-605.
Non-Patent Document 8: Wightman, L. et al., (2001) Different behavior of branched and linear polyethylenimine for gene delivery in vitro and in vivo. J. Gene Med. 3, 362-372.
Non-Patent Document 9: Durocher, Y. et al., (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res. 30, e9.
Non-Patent Document 10: Brissault, B. et al., (2003) Synthesis of linear polyethylenimine derivatives for DNA transfection. Bioconjug. Chem. 14, 581-587.
Non-Patent Document 11: Thomas, M. et al., (2005) Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung. Proc. Natl. Acad. Sci. USA 102, 5679-5684.
Non-Patent Document 12: Ogris, M. et al., (1998) The size of DNA/transferring-PEI complexes is an important factor for gene expression in cultured cells. Gene Ther. 5, 1425-1433.
Non-Patent Document 13: Tseng, W. C. et al., (2007) Trehalose enhances transgene expression mediated by DNA-PEI complexes. Biotechnol. Prog. 23, 1297-1304.